Effects of Decamethylcyclopentasiloxane on Reproductive Systems in Female Rats

The female reproductive system becomes fertile through the action of hormones involved in the hypothalamic-pituitary-ovarian axis. On the other hand, estrogen-like endocrine disruptors released into the environment come into contact with humans by various routes and affect the reproductive system. Exposure to these chemicals can cause problems with the reproductive process, from egg ovulation to implantation, or cause female reproductive diseases. These reproductive problems cause infertility. Decamethylcyclopentasiloxane (D5) is used for lubrication in silicone polymers, households, and personal care products. In the case of D5, it is discharged through factory wastewater and can bioaccumulate. Therefore, it accumulates in the human body. In this study, D5 was administered orally for four weeks to determine the effects of D5 on the reproductive process. As a result, D5 increases the number of follicles in the ovary and suppresses the expression of genes related to the growth of follicles. In addition, it increases the gonadotropin hormone, inducing estradiol enhancement and progesterone reduction. Because of these changes in the reproductive system when exposed to D5, the industry should reconsider using D5.


Introduction
The female reproductive system becomes fertile through the action of various hormones involved in the hypothalamic-pituitary-ovarian (HPO) axis [1]. The action of the gonadotropin-releasing hormone secreted by the hypothalamus stimulates the pituitary gland to promote the secretion of the luteinizing hormone (LH) and the follicle-stimulating hormone (FSH), which are types of the gonadotropin hormone (GTH) [2]. GTH acts on the ovary to increase the secretion of steroid hormones, including estradiol (E2) and progesterone (P4) [3]. Enhanced steroid hormones inhibit the activity of the hypothalamus and pituitary gland [4].
The role of hormones in reproduction is very important, and changes in the HPO axis are caused by hormones produced in vivo. The types of exogenous hormones in the environment are diverse and have several effects. In the case of chemicals used mainly in factory processes, even if the product is not directly used, it is discarded into the environment through wastewater and exposed to humans [5][6][7]. Therefore, various chemicals present in the environment have biomagnification and accumulate in top predators [8][9][10].
Among the endocrine-disrupting chemicals (EDC), estrogenic chemicals cause direct damage to female reproductive organs [11,12]. The effects can cause disease of the reproductive organs, leading to infertility, such as polycystic ovary syndrome (PCOS) and endometriosis (EMS). PCOS is a common disease affecting 5 to 18% of women worldwide. This causes symptoms, such as an excess of androgens, anovulation, and polycystic ovaries, which lead to various complications [13,14]. EMS is also a disease in many women, in which the endometrium attaches to the extrauterine tissue and proliferates. This symptom deforms the pelvic structure and causes pain due to adhesion to a wide range of tissues [15,16].

Embryoid Body Test (EBT)
The cell viability was measured by seeding 800 cells in 50 µL/well of maintenance media into a 96-well plate. D5 was treated at a concentration of 10 −3 -10 −11 M so that the volume per well was 200 µL. After culturing for three days, the plate was washed twice with D-PBS and 10 µL of EZ-cytox (EZ-500, Dogenbio, Seoul, Republic of Korea), and each well was treated with 90 µL of media. The plate was incubated for 1 h, and the absorbance at a wavelength of 450 nm was measured.
EB formation was performed using the hanging drop method. In a 90 mm petri dish, up to 85 drops of differentiated media were added, including 800 cells in each 20 µL drop. The samples were cultured for 3 days and collected in a 35 mm petri dish. Images were taken at 100× magnification with a phase contrast microscope (IX71, Olympus, Tokyo, Japan). For the eight images obtained, the size of the EB area was measured using Image J (NIH, Bethesda, MD, USA).

Tissue Fixation and Section
Immediately after obtaining the ovary tissue, it was fixed in 10% neutral buffered formalin and embedded in paraffin. A total of 5 serial sections were performed in units of 60 µm. The sections (6 µm thick) were stained with hematoxylin and eosin.

RNA Extraction, Complementary DNA Synthesis, and Quantitative Real-Time PCR
For total RNA extraction, the obtained tissue was placed in containing beads Trizol (15596026, Invitrogen, Waltham, MA, USA) and homogenized using bead ruptor 12 (OMNI, Kennesaw, GA, USA). Chloroform was added at one-fifth of the trizol level and centrifuged at 14,000 rpm for 10 min. The supernatant was obtained by adding 100% ethanol at 1.5 times the volume of the supernatant and transferred to a spin column. The mixture was centrifuged at 8000 rpm for 15 s. RW1 buffer (700 µL, 1053394, Qiagen, Hilden, Germany) was added and centrifuged. RPE buffer (350 µL, 1018013, Qiagen, Hilden, Germany) was added, and the centrifugation was run twice. The membrane was dried by running the spin column at 14,000 rpm for 1 min. For elution, 100 µL of DNase RNase-free water was added to the membrane. After 30 s, elution proceeded at 14,000 rpm for 1 min.
A Quantstudio 3 real-time PCR instrument (applied biosystems, Foster City, CA, USA) was used to measure the quantitative real-time PCR (qPCR). In a qPCR cocktail, 6.25 µL of 2× prime q-master mix (with SYBR green I), 0.25 µL of 50× ROX dye (Q-9212, GENETBIO, Daejeon, Republic of Korea), 1 µL of forward and reverse primer (200 µM each), 3 µL of distilled water, and 2 µL of cDNA were included. Table 1 lists the primer information. The qPCR cycle consisted of 40 cycles, followed by 95 • C for 30 s for denaturation, 58 • C for 30 s for annealing, and 72 • C for 30 s for elongation. The fluorescence was measured at the end of each PCR stage. The ∆∆CT values were analyzed using the Quantstudio design and analysis software. Table 1. qPCR primer information.

Blood Collection and Serum Separation
For serum biochemical analysis, blood was collected by cardiac puncture after sacrifice. The blood was incubated overnight at 4 • C and centrifuged at 4000× g for 10 min. The serum was immediately stored in a −70 • C deep freezer. The serum concentrations of LH (CEA441Ra), FSH (CEA830Ra), and AMH (CEA228Ra) were measured using a Cloud-clone (Katy, TX, USA) ELISA kit.

Statistical Analyses
The data were visualized and analyzed statistically using GraphPad Prism 8 for Windows (GraphPad Software, San Diego, CA, USA). The values in the graph were reported as means ± standard deviations (SD). An ordinary one-way analysis of variance (ANOVA) was used for comparison, and statistical significance was considered p-value < 0.05.

Developmental Toxicity of D5
When treated with D5, the cell viability of the two cell lines, mES and 3T3-L1, had IC 50 values of 0.04381 µM (mES) and 0.5742 µM (3T3), respectively ( Figure 1A-D). In addition, the half inhibitory concentration of the EB size, ID 50 , was 0.001663 µM ( Figure 1C,D). A value of 4.803 was obtained by substituting this derived value into the previously developed discriminant function ( Figure 1D). D5 had developmental toxicity because the discriminant function score was higher than the reference value of −0.667. 1C,D). A value of 4.803 was obtained by substituting this derived value into the previously developed discriminant function ( Figure 1D). D5 had developmental toxicity because the discriminant function score was higher than the reference value of −0.667.

Polycystic Ovarian Morphology Induced by D5
The number of follicles located at two growth stages was measured to confirm follicle development in the ovary. Upon treatment with D5, the number of early follicles, including primordial, primary, and secondary follicles, increased in the 200 mg/kg group ( Figure  2A). In addition, the number of late follicles, including antral and preovulation follicles, increased in the 100 mg/kg and 200 mg/kg groups ( Figure 2B). Therefore, the total number of follicles increased in the 100 mg/kg and 200 mg/kg groups ( Figure 2C). This confirmed that the number of follicles in the ovary increased when exposed to D5. An increase in follicles in the ovary also means a representative phenotype of PCOS, and ovulation is not taking place due to the accumulation of follicles.

Polycystic Ovarian Morphology Induced by D5
The number of follicles located at two growth stages was measured to confirm follicle development in the ovary. Upon treatment with D5, the number of early follicles, including primordial, primary, and secondary follicles, increased in the 200 mg/kg group ( Figure 2A). In addition, the number of late follicles, including antral and preovulation follicles, increased in the 100 mg/kg and 200 mg/kg groups ( Figure 2B). Therefore, the total number of follicles increased in the 100 mg/kg and 200 mg/kg groups ( Figure 2C). This confirmed that the number of follicles in the ovary increased when exposed to D5. An increase in follicles in the ovary also means a representative phenotype of PCOS, and ovulation is not taking place due to the accumulation of follicles. 1C,D). A value of 4.803 was obtained by substituting this derived value into the previously developed discriminant function ( Figure 1D). D5 had developmental toxicity because the discriminant function score was higher than the reference value of −0.667.

Polycystic Ovarian Morphology Induced by D5
The number of follicles located at two growth stages was measured to confirm follicle development in the ovary. Upon treatment with D5, the number of early follicles, including primordial, primary, and secondary follicles, increased in the 200 mg/kg group ( Figure  2A). In addition, the number of late follicles, including antral and preovulation follicles, increased in the 100 mg/kg and 200 mg/kg groups ( Figure 2B). Therefore, the total number of follicles increased in the 100 mg/kg and 200 mg/kg groups ( Figure 2C). This confirmed that the number of follicles in the ovary increased when exposed to D5. An increase in follicles in the ovary also means a representative phenotype of PCOS, and ovulation is not taking place due to the accumulation of follicles.

Weight of Uterus and Ovary Compared to Body Weight
The body weight was measured, and the % body weight of the ovary and uterus was checked to confirm the induction of obesity in rats exposed to D5. No significant change in Toxics 2023, 11, 302 6 of 13 body weight ( Figure 3A) and ovary and uterus weight ( Figure 3B,C) were observed. There was no change in organ weight when exposed to D5, suggesting that it did not induce obesity. The body weight was measured, and the % body weight of the ovary and uterus was checked to confirm the induction of obesity in rats exposed to D5. No significant change in body weight ( Figure 3A) and ovary and uterus weight ( Figure 3B,C) were observed. There was no change in organ weight when exposed to D5, suggesting that it did not induce obesity.

Changes in Gonadotropin Hormones and Anti-Müllerian Hormone
The serum hormone concentrations were checked to investigate the cause of the increased follicle formation in the ovary. When exposed to D5, there was no significant change in FSH, a hormone that stimulates follicle formation in the serum ( Figure 4A). AMH, a marker of PCOS, also showed no significant change ( Figure 4B). On the other hand, there was an increase in LH in the 100 mg/kg and 200 mg/kg groups ( Figure 4C). The LH/FSH ratio, another known marker of PCOS, increased ( Figure 4D). The possibility of affecting the target organ was confirmed by changing the hormone concentration. Through the increase in LH in rats exposed to D5, D5 affected the pituitary gland and increased GTH secretion.

Changes in Gonadotropin Hormones and Anti-Müllerian Hormone
The serum hormone concentrations were checked to investigate the cause of the increased follicle formation in the ovary. When exposed to D5, there was no significant change in FSH, a hormone that stimulates follicle formation in the serum ( Figure 4A). AMH, a marker of PCOS, also showed no significant change ( Figure 4B). On the other hand, there was an increase in LH in the 100 mg/kg and 200 mg/kg groups ( Figure 4C). The LH/FSH ratio, another known marker of PCOS, increased ( Figure 4D). The possibility of affecting the target organ was confirmed by changing the hormone concentration. Through the increase in LH in rats exposed to D5, D5 affected the pituitary gland and increased GTH secretion.

Weight of Uterus and Ovary Compared to Body Weight
The body weight was measured, and the % body weight of the ovary and uterus was checked to confirm the induction of obesity in rats exposed to D5. No significant change in body weight ( Figure 3A) and ovary and uterus weight ( Figure 3B,C) were observed. There was no change in organ weight when exposed to D5, suggesting that it did not induce obesity.

Changes in Gonadotropin Hormones and Anti-Müllerian Hormone
The serum hormone concentrations were checked to investigate the cause of the increased follicle formation in the ovary. When exposed to D5, there was no significant change in FSH, a hormone that stimulates follicle formation in the serum ( Figure 4A). AMH, a marker of PCOS, also showed no significant change ( Figure 4B). On the other hand, there was an increase in LH in the 100 mg/kg and 200 mg/kg groups ( Figure 4C). The LH/FSH ratio, another known marker of PCOS, increased ( Figure 4D). The possibility of affecting the target organ was confirmed by changing the hormone concentration. Through the increase in LH in rats exposed to D5, D5 affected the pituitary gland and increased GTH secretion.

Changes in Steroid Hormones
The secretion of steroid hormones was changed by the increased secretion of LH. Exposure to D5 increased serum E2 concentrations ( Figure 5A). On the other hand, the concentration of P4 did not change ( Figure 5B), and the concentration of testosterone increased ( Figure 5C). As a result, all steroid hormones increased due to the increase in

Changes in Steroid Hormones
The secretion of steroid hormones was changed by the increased secretion of LH. Exposure to D5 increased serum E2 concentrations ( Figure 5A). On the other hand, the concentration of P4 did not change ( Figure 5B), and the concentration of testosterone increased ( Figure 5C). As a result, all steroid hormones increased due to the increase in GTH, and it was necessary to confirm what problems occurred in the organs of the female reproductive system.

Expression Levels of the Gonadotropin Hormone Receptor and the Steroid Hormone Receptor in Reproductive Organs
As in the previous results, receptor expression also increased with GTH. Steroid hormones act mainly by binding to receptors. Therefore, the changes in the expression level of steroid hormone receptors were observed when exposed to D5. First, there was no change in the levels of Pgr, Esr1, and Esr2 expression in the ovary ( Figure 6A-C). The expression levels of Fshr ( Figure 6D) and Lhcgr were also increased ( Figure 6E). Second, there was no significant change in Pgr in the uterus ( Figure 6F), and the levels of Esr1 and Esr2 expression decreased ( Figure 6G,H). As a result, the effect of Fshr and Lhcgr in the ovary increased, and the effect of E2 targeting the uterus increased.

Expression Levels of the Gonadotropin Hormone Receptor and the Steroid Hormone Receptor in Reproductive Organs
As in the previous results, receptor expression also increased with GTH. Steroid hormones act mainly by binding to receptors. Therefore, the changes in the expression level of steroid hormone receptors were observed when exposed to D5. First, there was no change in the levels of Pgr, Esr1, and Esr2 expression in the ovary ( Figure 6A-C). The expression levels of Fshr ( Figure 6D) and Lhcgr were also increased ( Figure 6E). Second, there was no significant change in Pgr in the uterus ( Figure 6F), and the levels of Esr1 and Esr2 expression decreased ( Figure 6G,H). As a result, the effect of Fshr and Lhcgr in the ovary increased, and the effect of E2 targeting the uterus increased.

Comparison of Gene Expression in Ovary
3.5.1. Steroidogenesis Gene Figure 6 shows the increased effect of steroid hormone, and changes in steroidogenesis in the ovary were checked to find the cause of their increased effect. The changes in the genes that play important roles in synthesizing steroid hormones were identified. The level of Star expression was increased in all groups treated with D5 ( Figure 7A). On the other hand, the level of Cyp11a1 expression decreased ( Figure 7B), and there was no significant change in the level of Hsd3b1 expression ( Figure 7C). Cyp19a1 showed a sharply increasing pattern in the 10 mg/kg group ( Figure 7D). Therefore, the amount of steroid hormone synthesis increased, and the conversion to E2 also increased.

Folliculogenesis Gene
The steroid hormone is produced in the ovary and affects the hypothalamus, pituitary gland, ovary, and uterus. The cause of histological changes in the ovary was examined by measuring the expression levels of genes that play an important role in follicle formation. First, in the case of Amh, the expression level increased effectively ( Figure 8A), and Sohlh2, Foxl2, and Kitlg showed a decreasing pattern ( Figure 8B-D). As a result, the ovaries exposed to D5 had developmental problems and did not ovulate normally. Anovulation in this ovary causes menstrual irregularities and infertility.

Comparison of Gene Expression in Ovary
3.5.1. Steroidogenesis Gene Figure 6 shows the increased effect of steroid hormone, and changes in steroidogenesis in the ovary were checked to find the cause of their increased effect. The changes in the genes that play important roles in synthesizing steroid hormones were identified. The level of Star expression was increased in all groups treated with D5 ( Figure 7A). On the other hand, the level of Cyp11a1 expression decreased ( Figure 7B), and there was no significant change in the level of Hsd3b1 expression ( Figure 7C). Cyp19a1 showed a sharply increasing pattern in the 10 mg/kg group ( Figure 7D). Therefore, the amount of steroid hormone synthesis increased, and the conversion to E2 also increased.

Folliculogenesis Gene
The steroid hormone is produced in the ovary and affects the hypothalamus, pituitary gland, ovary, and uterus. The cause of histological changes in the ovary was examined by measuring the expression levels of genes that play an important role in follicle formation. First, in the case of Amh, the expression level increased effectively ( Figure 8A), and Sohlh2, Foxl2, and Kitlg showed a decreasing pattern ( Figure 8B-D). As a result, the ovaries exposed to D5 had developmental problems and did not ovulate normally. Anovulation in this ovary causes menstrual irregularities and infertility. The steroid hormone is produced in the ovary and affects the hypothalamus, pitui-tary gland, ovary, and uterus. The cause of histological changes in the ovary was examined by measuring the expression levels of genes that play an important role in follicle formation. First, in the case of Amh, the expression level increased effectively ( Figure 8A), and Sohlh2, Foxl2, and Kitlg showed a decreasing pattern ( Figure 8B-D). As a result, the ovaries exposed to D5 had developmental problems and did not ovulate normally. Anovulation in this ovary causes menstrual irregularities and infertility.

Confirmation of Implantation-Related Gene Expression in the Uterus
The steroid hormones secreted from the ovary influence the target organ, which is the uterus. The expression levels of the genes related to the sensitivity of uterine epithelium were confirmed. The levels of Hand2, Fkbp4, and Hoxa10 expression decreased (Figure 9A-C). Hence, D5 can cause problems in the implantation process. Similar to anovulation, this can cause infertility.

Confirmation of Implantation-Related Gene Expression in the Uterus
The steroid hormones secreted from the ovary influence the target organ, which is the uterus. The expression levels of the genes related to the sensitivity of uterine epithelium were confirmed. The levels of Hand2, Fkbp4, and Hoxa10 expression decreased ( Figure 9A-C). Hence, D5 can cause problems in the implantation process. Similar to anovulation, this can cause infertility.

Discussion
D5 is used in cosmetics and household products and is discharged to wastewater during manufacturing. The main cosmetics users are women, who are more exposed to D5 than men. Therefore, the effects of D5 on women were confirmed. A previous study reported that D4, which has a similar structure to D5, affects the female reproductive system [34][35][36]. Because of the similar structure, D5 was also studied with the expectation that it would influence the female reproductive system. Reproduction proceeds in the order of egg ovulation, fertilization, and implantation [37]. Among them, the effects of D5 on ovulation and implantation were studied.
When radioactively labeled D5 is administered orally, it is excreted in the urine of rats as various metabolites [38]. In another study, when two concentrations of D5 were administered orally, urinary recovery was 4.39% and 8.15% for the high and low doses, respectively [39]. This means that D5 is saturated in the metabolic process in the body. Therefore, this study focused on the effects of D5 rather than the effect of the concentration of D5.

Discussion
D5 is used in cosmetics and household products and is discharged to wastewater during manufacturing. The main cosmetics users are women, who are more exposed to D5 than men. Therefore, the effects of D5 on women were confirmed. A previous study reported that D4, which has a similar structure to D5, affects the female reproductive system [34][35][36]. Because of the similar structure, D5 was also studied with the expectation that it would influence the female reproductive system. Reproduction proceeds in the order of egg ovulation, fertilization, and implantation [37]. Among them, the effects of D5 on ovulation and implantation were studied.
When radioactively labeled D5 is administered orally, it is excreted in the urine of rats as various metabolites [38]. In another study, when two concentrations of D5 were administered orally, urinary recovery was 4.39% and 8.15% for the high and low doses, respectively [39]. This means that D5 is saturated in the metabolic process in the body. Therefore, this study focused on the effects of D5 rather than the effect of the concentration of D5.
The egg is ovulated through follicle development. At this time, various transcription factors act on the growth of the oocyte. Kitlg is in early oocytes and granulosa cells and affects follicle development [40]. Kitlg and c-KIT signaling are essential for oocyte survival, and they activate the PI3K pathway when combined [41]. Activation of PI3K means an increase in cell proliferation [42]. Moreover, Foxl2 is involved in most ovarian development and function in granulosa cells [43]. Defective Foxl2 stops the squamous to the cuboidal transition of granulosa cells, preventing secondary follicle development [44]. In addition, Sohlh2 was expressed predominantly in early primordial and primary follicles. A deficiency of this gene caused oocyte and follicle loss [45]. In this study, Kitlg and Foxl2 were decreased by the D5 treatment, which inhibited granulosa cell differentiation, indicating that problems occurred in early follicle development. In addition, the decrease in Sohlh2 indicates that D5 can cause the premature depletion of oocytes.
A typical egg is fertilized and implanted into the endometrium. Among the several genes contributing to endometrial receptivity, Hand2 suppresses several fibroblast growth factors to increase receptivity in the uterus [46]. The inhibition of Hand2 causes a decrease in endometrial receptivity caused by abnormal endometrial proliferation [47]. Hoxa10 also plays an important role in the implantation, decidualization, and immune regulation of the adult uterus. Low expression of Hoxa10 impairs endometrial differentiation [48]. In addition, Fkbp4 must bind to Pgr for transcript activation during implantation and is a downstream gene of Hoxa10 [49,50]. Endometrium proliferation decreased when the expression of Hand2, Hoxa10, and Fkbp4 was reduced in rats expressed to D5. These problems lead to decreased endometrial receptivity, resulting in infertility.
The problems with the reproduction process may have been caused by dysfunction of the HPO axis. An increase in LH causes problems with follicle maturation and induces abnormal reproductive processes [51]. The resulting increase in the LH/FSH ratio also means an increase in androgen production in theca cells [52]. Exposure to D5 caused problems with follicle maturation and affected ovarian development by stimulating corpus luteum formation.
The secreted hormones combine with hormone receptors and are activated [53]. Therefore, the change in the receptor expression level indicates the action of the hormone. The Lhcgr gene, an LH receptor, is expressed in granulosa cells [54]. In addition, the FSH receptor, Fshr, is a G-protein coupled receptor located in the granulosa cells and controls the processing and movement of steroid hormones [55]. Therefore, the increase in Lhcgr and Fshr confirmed the increase in steroid hormone production in ovary granulosa cells caused by GTH.
The steroid hormone is synthesized in the ovary, and failure or overexpression of this process significantly affects the reproductive system. Steroidogenesis is a step-by-step process. Star promotes the transport of cholesterol within the mitochondria [56]. The transported cholesterol is converted to pregnenolone by Cyp11a1 [57]. The converted pregnenolone is metabolized by Hsd3b1 to synthesize P4 [58]. Cyp19a1 also converts testosterone to E2 [59]. Through this, Star and Cyp19a1 induction increased E2 expression and decreased Cyp11a1 reduced P4 synthesis. Hence, D5 causes problems in steroidogenesis in the ovary.
Esr expression increased and Pgr expression decreased when pituitary gland cells were exposed to E2 [60]. Changes in the expression level of these steroid hormone receptors could indirectly confirm the effect of E2. Through the above results and changes in steroid hormone receptors, when exposed to D5, the effect of E2 in the body increased due to the decrease in Esr expression in the uterus. Increased expression of steroid hormones causes a variety of problems. Increased E2 causes endometriosis, an estrogen-dependent disease, or increases the risk of breast, uterine, and ovarian cancer. In addition, the increased secretion of testosterone in women increases the incidence of polycystic ovary syndrome.
In addition, developmental toxicity was checked to determine if there were any problems with embryo growth after egg implantation.
The experiment found that exposure to D5 had several negative effects, including increasing GTH levels, decreasing follicle development, and increasing both the number of follicles and steroid hormone expression in the ovary. This study confirmed that exposure to D5 reduces endometrial receptivity and has developmental toxicity. These results suggest that D5 has similar effects to E2, a hormone essential for female reproductive health. These findings imply that D5 exposure disrupts the normal balance of steroid hormones in the body, potentially leading to various health problems. Thus, further discussion is necessary to determine the safety of D5 in products for women and pregnant women by industry.

Conclusions
Exposure to D5 in the female body increases the number of follicles in the ovary, increases the secretion of LH in the serum and the steroid hormones estradiol and testosterone, and indicates fetal developmental disorders. The limitations of the use of D5 should be considered because there are still many industries that use it.